The mammalian immune system has evolved a variety of mechanisms to protect the host from microorganisms, an important component of this response being mediated by cells referred to as T cells and by antibodies derived from B cells. In combating bacterial infections, antibodies are especially important but likewise are specialized T cells that function primarily by recognizing and killing infected cells. The latter also function by secreting soluble molecules called cytokines that mediate a variety of functions of the immune system. Thus, the immune system is highly developed to deal with infectious organisms as well as with the elimination of cells infected with such organisms. Among the latter are viral infections, such as HBV infection.
Hepatitis B virus (HBV) is a member of the Hepadnaviridae family of viruses which also includes woodchuck hepatitis virus (WHV) and duck hepatitis B virus. These viruses are primarily hepatotropic with infections characterized by fever, fatigue, muscle aches, and yellowing of the eyes and/or skin. The severity of these symptoms can vary with a proportion of cases being asymptomatic. More than 2.5 billion people worldwide have been infected by HBV, but for the vast majority of adults encountering the virus (>90%), the infection is acute and readily cleared by the immune system. For the remaining 5-10% of adults, and for neonates and unvaccinated children, HBV establishes a chronic infection. Approximately 370 million people worldwide are chronically infected and over 500,000 people die each year due to complications from HBV. These complications include liver cirrhosis, liver failure, and/or hepatocellular carcinoma (HCC) and it is estimated that up to 40% of chronically infected patients will develop at least one of these complications.
The primary determinant of whether hepatitis B virus is cleared or establishes a chronic infection is the robustness of the immune response, in particular the CD8+ T cell response. Data from both animal models and infected patients indicate that strong innate immune responses are crucial in controlling initial HBV replication and for subsequently activating the adaptive T cell response (reviewed in Rehermann and Nascimbeni; Bertoletti and Gehring). In patients that resolve acute infections, there are greater numbers of IFN-γ secreting CD4+ and CD8+ T cells with a broader range of epitope recognition than in chronically infected patients (as reviewed in Bertoletti and Gehring; Desmond et al.). Although individuals that initially fail to mount vigorous T cell responses develop chronic infection, data indicate that virus specific T cells are still capable of a broad, effective T cell response. Rehermann et al. demonstrated that a small number of chronically infected individuals mount robust CTL responses against HBV either spontaneously or in response to IFN-α treatment. These T cells are directed against multiple proteins indicating that chronically infected patients can also mount a broad response to viral antigens. These data suggest that therapeutic interventions designed to stimulate robust and multi-epitope specific responses may be sufficient to resolve chronic HBV infections. Yet, despite an effective prophylactic vaccine, there are currently no therapies capable of eliminating HBV from chronically infected individuals. A number of anti-HBV therapeutic vaccines have been tested including traditional prophylactic vaccines, antigen/antibody complexes, lipopeptide, DNA, and recombinant virus based strategies with limited success. Thus, there is a critical need for more targeted therapeutic vaccines capable of inducing robust, sustained T cell responses capable of permanent clearance of virus.
Therapeutic peptide based vaccines are an attractive method for inducing CD4+ and CD8+ T cell responses in chronically infected individuals. While other vaccine formulations (such as DNA and recombinant virus vaccines) induce T cell responses, the peptide epitopes generated after vaccination may not accurately reflect those generated during chronic infection and therefore may not induce the necessary polyclonal response needed to clear the virus. In contrast, formulating a vaccine with multiple epitopes presented by the chronically infected cells that are capable of activating T cells to generate a polyclonal response would bypass the need for translation and processing of the parent protein and allow for expansion of the appropriate T cell specificities.
Peptide antigens for these early stage clinical studies were identified by motif prediction algorithms and selected by screening CTLs from acute and chronically HBV infected patients. However, the T cell epitopes presented by HBV infected cells have not been reported or used in a clinical study.
Here we took an immunoproteomic approach to identify MHC class I peptides presented by chronic HBV infected cells. This approach has distinct advantages over traditional vaccine design algorithms as it identifies antigens naturally processed and presented by infected, but not healthy, cells. The identification of peptides and proteins derived from HBV infection that are effectively recognized by the cellular arm of the immune response forms the basis for a new and effective vaccine. Such peptides are displayed on the surface of infected cells in association with MHC class I and class II molecules and serve as recognition targets for cytolytic and helper T cells of the immune system.
The present disclosure involves peptides that are associated with the HLA-A2, and HLA-A24 molecules, HLA-A2 supertypes, and HLA-A24 supertypes. A supertype is a group of HLA molecules that present at least one shared epitope. The present disclosure involves peptides that are associated with HLA molecules, and with the genes and proteins from which these peptides are derived.
Several methods have been developed to identify the peptides recognized by CTL, each method relying on the ability of a CTL to recognize and kill only those cells expressing the appropriate class I MHC molecule with the peptide bound to it. Such peptides can be derived from a non-self source, such as a pathogen (for example, following the infection of a cell by a virus, such as HBV virus infection) or from a self-derived protein within a cell, such as a cancerous cell.
Three different methodologies have typically been used for identifying the peptides that are recognized by CTLs in infectious disease field. These are: (1) the genetic method; (2) motif analysis; (3) the immunological and analytical chemistry methods or the Immunoproteomics method. The genetic and motif prediction methodologies have typically been used for identifying the peptides that are recognized by CTLs, which suffer from various drawbacks. A useful technique has been the immunoproteomics method involving a combination of cellular immunology and mass spectrometry. This approach involves the actual identification of endogenous CTL epitopes present on the cell surface by sequencing the naturally occurring peptides associated with class I MHC molecules. In this approach, cells are first lysed in a detergent solution, the peptides associated with the class I MHC molecules are purified, and the peptides are fractionated by high performance liquid chromatography (HPLC). Peptide sequencing is readily performed by tandem mass spectrometry. The sequence can be confirmed by direct synthesis thereof (See Examples 4 and 5, below). Once confirmed such synthetic peptides can be used to test their ability to activate CTLs against cells infected with the HBV virus.
A number of recent reports for different types of virus infections provide evidence that CTL specific for epitopes that are naturally processed and presented by infected cells have markedly greater impact on the control of virus replication. Undoubtedly, CTLs have been shown to play an important role in the elimination of HBV-infected cells. Thus, identification of antigenic peptides that are presented by infected cells and recognized by epitope-specific CTLs may suggest new ways to suppress viral replication and prevent persistent infection. Multiple peptides from conserved regions of HBV may prove essential in the development of a universally immunogenic vaccine.
Little is known about cross genotypes conserved T cell epitopes that are immunologically relevant in eliciting an effective T cell response to the various HBV genotypes. Several groups have attempted to identify T cell epitopes by either motif prediction of MHC binding peptides from HBV proteins, or by screening overlapping peptides from structural and nonstructural viral proteins. Screening PBMCs from infected individuals using a panel of algorithm-derived peptide sequences identified a few cross genotype specific T cell epitopes. However, a comprehensive analysis of naturally presented epitopes on infected cells has never been undertaken or reported.
Immunization with virus-derived, class I MHC-encoded molecule-associated peptides, or with a precursor polypeptide or protein that contains the peptide, or with a gene that encodes a polypeptide or protein containing the peptide, are forms of immunotherapy that can be employed in the treatment of infections. These forms of immunotherapy require that immunogens be identified so that they can be formulated into an appropriate vaccine.